Scleroderma or Systemic Sclerosis (SSc) is a disease of unknown etiology characterized by the excessive deposition of collagen and other connective tissue components in skin and multiple internal organs, prominent and often severe alterations in the microvasculature, and numerous humoral and cellular immunologic abnormalities. Although the mechanisms involved in the pathogenesis of SSc are not completely known, it is clear that cutaneous, visceral, and vascular fibrosis is responsible for the most severe clinical manifestations and the mortality of the disease. The overall objective of this application is to identify some of the molecular events that may be involved in the exaggerated production of collagen and in the microvascular abnormalities in SSc. Several novel areas of investigation have been identified during the previous funding period including the significant association of SSc with certain alleles of the gene encoding Allograft InflammatoryFactor 1 (AIF1), the demonstration of ATP1 in inflammatory and endothelial cells in SSc affected microvasculature,the demonstration of a possible role of geranylgeranyl isoprenylation and of RhoA and protein kinase C8 (PKC8) in the upregulated expression of collagen genes in SSc, and the demonstration that statins exert potent inhibition of collagen gene expression. Based on these results, we propose to test the following hypotheses: (1) AIF1, a protein expressed in lymphocytes, macrophages and microvascular fibroproliferative lesions from SSc patients,plays an important role in the pathogenesis of the vascular abnormalities characteristic of the disease; (2) the excessive production of collagen in SSc involves abnormalities in non-Smad pathways including abnormalities in RhoA signaling, geranylgeranyl isoprenylation and in the activation of the PKC5 pathway. These abnormalities result in the activation of transcription factors which eventually stimulate the expression of regulatory elements in the collagen genes. To test these hypotheses, we will pursue the following Specific Aims: Specific Aim 1: Determination of the role of AIF1 in the pathogenesis of the vascular obliterative process in SSc and further characterization of the functional role of the protein. Specific Aim 2: Examination of alterations in non-Smad pathways of collagen gene expression regulation focusing on the identification of abnormalities in RhoA, geranylgeranyl, and PKC8-mediated steps of collagen gene expression regulation in SSc fibroblasts. It is expected that results from these studies will provide valuable clues towards the understanding of the pathogenesis of tissue fibrosis and of the prominent microvascular alterations in SSc and may open new avenues of investigation towards the development of therapeutic agents for this serious and incurable disease.